Methods for controlling fucosylation levels in proteins

ABSTRACT

The present invention relates to a method or process for controlling, inhibiting or reducing protein fucosylation in a eukaryote and/or a eukaryotic protein expression system. Said method comprises carrying out the protein expression and/or post-translational modification in the presence of an elevated total concentration of manganese or manganese ions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of and the priority toprovisional Indian Patent applications 3262/CHE/2013 filed on 23 Jul.2013 and 3265/CHE/2013 filed on 23 Jul. 2013 with the Indian PatentOffice. The content of the said applications filed on 23 Jul. 2013 isincorporated herein by reference for all purposes in its entirety,including an incorporation of any element or part of the decision,claims or drawings not contained herein and referred to in Rule 20.5(a)of PCT, pursuant to Rule 4.18 of the PCT.

TECHNICAL FIELD

The present invention relates to methods for controlling fucosylationlevels in proteins.

BACKGROUND OF THE DISCLOSURE

Proteins expressed in eukaryotic expression systems undergo a process ofpost-translational modification, which involves glycosylation.Eukaryotic expression systems which have been established today for theproduction of glycoproteins, like IgG and other monoclonal antibodiescomprising an Fc region add N-glycans to the polypeptide chains.

In IgG, the most important N-glycan is bound at Asn 297 of both CH2chains (see FIG. 14), which comprises, among others, N-acetyl-neuraminicacid (sialic acid), N-acetyl-glucosamine, galactose, mannose, and fucoseresidues.

This applies, basically, for transgenic plant expression systems as wellas for mammalian cell lines, insect cell lines etc. In all these cases,the N-glycan comprises at least one fucose residue which is bound eitherα-3-glycosidically or α-6-glycosidically to the N-acetyl-glucosamineresidue bound to the Asn residue of the polypeptide chain.

Yeast expression systems tend to produce hyperglycoproteins rich inmannose, which often lead to unwanted immune reactions when thetherapeutic antibody is administered to a patient. Baculovirustransfected insect cell systems cause problems due to hypoglycosylation,which negatively affects the effector function of therapeuticantibodies. Furthermore, the major disadvantage are the catalyticproperties of infectious baculovirus that narrows the window for fullIgG production.

ADCC is a mechanism of cell-mediated immunity whereby an effector cellof the immune system actively lyses a target cell that has been bound byspecific antibodies. It is one of the mechanisms through whichantibodies, as part of the humoral immune response, can act to limit andcontain infection. Classical ADCC-mediating effector cells are naturalkiller (NK) cells; but monocytes and eosinophils can also mediate ADCC.ADCC is part of the adaptive immune response due to its dependence on aprior antibody response.

Therapeutic antibodies which are used to elicit an ADCC in target cellsneed an Fc region in order to be recognized by Fc gamma receptors of thesaid effector cells.

Recent studies have shown that monoclonal antibodies having a reducedamount of fucose in its glycosylation pattern exhibit much higherAntibody-Dependent Cellular Cytotoxicity (ADCC) activity as compared tofucosylated antibodies. Again, it is basically position Asn 297 where alack of fucose residues leads to the increased ADCC. The mechanismbehind the increased ADCC of a low/no-fucose Antibody seems to bemediated by an increased affinity of a so modified Fc region to FcγR,for example FcγIIIa (CD16), the major Fc receptor for ADCC in humanimmune effector cells (Shields et al, 2002).

Fucosylation is one of the most common modifications involvingoligosaccharides on glycoproteins or glycolipids. Fucosylation comprisesthe attachment of a fucose residue to N-glycans, O-glycans, andglycolipids. O-Fucosylation, a special type of fucosylation, is veryimportant for Notch signaling. The regulatory mechanisms forfucosylation are complicated. Many kinds of fucosyltransferases, theGDP-fucose synthesis pathway, and GDP-fucose transporter are involved inthe regulation of fucosylation.

Glycosylation is known to impact the effector functions of therapeuticmonoclonal antibodies. Among the various sugar residues in theoligosaccharide chain of an antibody, fucose is one of the key sugarsthat affects the antibody dependent cellular cytotoxicity (ADCC) inducedby the product.

Manipulation of cell culture parameters is often employed to controlgalactosylation and sialylation of an antibody. Control of fucosylationis majorly done by using FUT8 knock out cells and other gene silencingmodels through cell line engineering.

US20090208500 discloses the production of antibodies with reduced fucoseand improved Fc function by manipulation of FUT8 Knock out cells.

U.S. Pat. No. 7,972,810 discloses cell culturing methods and mediacontaining manganese that improve glycosylation or sialylation ofglycoproteins, including erythropoietin and analogs or derivativesthereof. According to the disclosure, manganese increases sialylationand site occupancy in case of O-linked and N-linked glycosylation (i.e.lower aglycosylated product) and also increases terminalgalactosylation.

Further, fucose content of monoclonal antibodies can be controlled byculture medium osmolality for high antibody-dependent cellularcytotoxicity (Konno et al. 2012)

Yet, there is a need for an efficient method of producing glycoproteinsin a desired cell line while controlling the fucose content of therecombinantly engineered antibodies without undergoing the laboriousprocess of creating a FUT8 gene knockout in a selected cell line eachtime.

EMBODIMENTS OF THE INVENTION

These objects are met with methods and means according to theindependent claims of the present invention. The dependent claims arerelated to preferred embodiments. It is yet to be understood that valueranges delimited by numerical values are to be understood to include thesaid delimiting values.

SUMMARY OF THE DISCLOSURE

Before the invention is described in detail, it is to be understood thatthis invention is not limited to the particular component parts of thedevices described or process steps of the methods described as suchdevices and methods may vary. It is also to be understood that theterminology used herein is for purposes of describing particularembodiments only, and is not intended to be limiting. It must be notedthat, as used in the specification and the appended claims, the singularforms “a,” “an” and “the” include singular and/or plural referentsunless the context clearly dictates otherwise. It is moreover to beunderstood that, in case parameter ranges are given which are delimitedby numeric values, the ranges are deemed to include these limitationvalues. According to one embodiment of the invention, a method orprocess for modifying fucosylation in a eukaryote and/or a eukaryoticprotein expression system is provided, in which method or process thetotal concentration of manganese or manganese ions in the medium iscontrolled.

Fucosylation of glycoproteins is accomplished by fucosyltransferases(FUT). These are enzymes that transfer an L-fucose sugar from aGDP-fucose (guanosine diphosphate-fucose) donor substrate to an acceptorsubstrate. The acceptor substrate can be another sugar such as thetransfer of a fucose to a core GlcNAc (N-acetylglucosamine) sugar as inthe case of N-linked glycosylation, or to a protein, as in the case ofO-linked glycosylation produced by O-fucosyltransferase. There arevarious fucosyltransferases in mammals, the vast majority of which, arelocated in the Golgi apparatus. The O-fucosyltransferases have recentlybeen shown to localize to the endoplasmic reticulum (ER). Examples ofmammalian fucosyltransferases are FUT1; FUT2; FUT3; FUT4; FUT5; FUT6;FUT7; FUT8; FUT9; FUT10 and FUT11.

Manganese is an essential trace element which participates in manyenzyme systems, although its role is not yet fully understood. It actsas a cofactor in enzymes that are essential for energy production and isinvolved in the metabolism of glucose, glycogen storage in the liver,protein digestion and synthesis of cholesterol and fatty acids. It isalso essential for the synthesis of DNA and RNA molecules.

Manganese is essential for the growth and maintenance of the nervoussystem, the development and maintenance of bones and joints, thefunction of female sex hormones and thyroid hormones. Superoxidedismutase (SOD, MnSOD) is an antioxidant enzyme that in its structurecontains manganese.

In extracellular liquids or Eukaryotes, manganese is practically absent,while in mammals, the intracellular concentration of Manganese is in therange of 0.010 picogram/cell-0.10 picogram/cell.

However, the inventors surprisingly found that that the concentration ofmanganese has a direct effect on the fucosylation level ofglycoproteins.

Thus, the present invention provides for modification of the fucosecontent of glycosylated proteins by varying the total concentration ofmanganese or manganese ions in media and feeds in the process.

Preferably, the method is a method or process to decrease fucosylation.In such method or process the protein expression and/orpost-translational modification is carried out in the presence of anelevated total concentration of manganese or manganese ions.

Surprisingly, the inventors found that under such conditions, theglycoproteins expressed have a decreased fucosylation level. Further,they found that the cell growth, viability and the titre of the proteinsproduced is not effected by the elevation of manganese or manganese ionconcentration.

Further, they found that other properties of the glycosylation pattern,namely G0 and Man5, are increased in the presence of an elevated totalconcentration of manganese or manganese ions.

As used herein, the term “fucosylation level” refers to the total amountof glycoproteins in which the glycans carry a fucose. Likewise, theterms “afucosylation level” and “% afucosylation” refers to thepercentage of glycoprotein which have no fucose in their glycans.

In a preferred embodiment of the method according to the invention, itis provided that the elevated concentration of manganese or manganeseions is in the range of ≧0.05 mM-≦10 mM.

Preferably, the elevated concentration of manganese or manganese ions is0.05; 0.1; 0.15; 0.2; 0.25; 0.3; 0.35; 0.4; 0.45; 0.5; 0.55; 0.6; 0.65;0.7; 0.75; 0.8; 0.85; 0.9; 0.95; 1; 1.05; 1.1; 1.15; 1.2; 1.25; 1.3;1.35; 1.4; 1.45; 1.5; 1.55; 1.6; 1.65; 1.7; 1.75; 1.8; 1.85; 1.9; 1.95;2; 2.05; 2.1; 2.15; 2.2; 2.25; 2.3; 2.35; 2.4; 2.45; 2.5; 2.55; 2.6;2.65; 2.7; 2.75; 2.8; 2.85; 2.9; 2.95; 3; 3.05; 3.1; 3.15; 3.2; 3.25;3.3; 3.35; 3.4; 3.45; 3.5; 3.55; 3.6; 3.65; 3.7; 3.75; 3.8; 3.85; 3.9;3.95; 4; 4.05; 4.1; 4.15; 4.2; 4.25; 4.3; 4.35; 4.4; 4.45; 4.5; 4.55;4.6; 4.65; 4.7; 4.75; 4.8; 4.85; 4.9; 4.95; 5; 5.05; 5.1; 5.15; 5.2;5.25; 5.3; 5.35; 5.4; 5.45; 5.5; 5.55; 5.6; 5.65; 5.7; 5.75; 5.8; 5.85;5.9; 5.95; 6; 6.05; 6.1; 6.15; 6.2; 6.25; 6.3; 6.35; 6.4; 6.45; 6.5;6.55; 6.6; 6.65; 6.7; 6.75; 6.8; 6.85; 6.9; 6.95; 7; 7.05; 7.1; 7.15;7.2; 7.25; 7.3; 7.35; 7.4; 7.45; 7.5; 7.55; 7.6; 7.65; 7.7; 7.75; 7.8;7.85; 7.9; 7.95; 8; 8.05; 8.1; 8.15; 8.2; 8.25; 8.3; 8.35; 8.4; 8.45;8.5; 8.55; 8.6; 8.65; 8.7; 8.75; 8.8; 8.85; 8.9; 8.95; 9; 9.05; 9.1;9.15; 9.2; 9.25; 9.3; 9.35; 9.4; 9.45; 9.5; 9.55; 9.6; 9.65; 9.7; 9.75;9.8; 9.85; 9.9; 9.95; or 10 mM.

These concentrations refer to the total concentration in the mediumwhere the protein expression and/or post-translational modificationtakes place. This means that, e.g., feed solutions can havesignificantly higher concentration of manganese or manganese ions.

Preferably, the concentration of manganese is accomplished by addingmanganese to the culture medium, and/or to the feed medium.

Likewise preferably, the manganese concentration is increased ordecreased during protein expression and/or post-translationalmodification.

In a preferred embodiment of the method according to the invention, itis provided that the protein expression and/or post-translationalmodification is carried out in a protein expression system selected fromthe group consisting of

Insect cellsFungal cellsYeast cellsProtozoan cells, and/orMammalian cells

Preferably, the mammalian cells are selected from the group consistingof murine cells (e.g, NS0), hamster cells (e.g., CHO or BHK) and/orhuman cells (e.g., PER.C6).

Preferably, the protein is a glycoprotein. More preferably, the proteinis a recombinant protein.

In a preferred embodiment of the method according to the invention, itis provided that the protein is an immunoligand.

The term “immunoligand” is used herein to mean an entity that has thecapability to bind to another biological entity with a sufficient degreeof sensitivity and/or specificity.

In another preferred embodiment of the method according to theinvention, it is provided that immunologand is at least one selectedfrom the group consisting of

-   a monoclonal antibody (murine, chimeric, humanized, human), or    derivative thereof-   a new antibody format-   a fusion peptide consisting of an immunoglobulin Fc region fused to    a target binding moiety, e.g, a receptor fragment

The above listed immunoligands comprise, preferably, an Fc region oranother domain that is capable of being glycosylated and/or binding toan Fc receptor, e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32),FcγRIIIA (CD16a), FcγRIIIB (CD16b).

As used herein, the term “monoclonal antibody (mAb)”, shall refer to anantibody composition having a homogenous antibody population, i.e., ahomogeneous population consisting of a whole immunoglobulin, or afragment or derivative thereof. Particularly preferred, such antibody isselected from the group consisting of IgG, IgD, IgE, IgA and/or IgM, ora fragment or derivative thereof.

As used herein, the term “derivative” shall refer to protein constructsbeing structurally different from, but still having some structuralrelationship to, the common antibody concept.

Methods for the production and/or selection of chimeric, humanisedand/or human mAbs are known in the art. For example, U.S. Pat. No.6,331,415 by Genentech describes the production of chimeric antibodies,while U.S. Pat. No. 6,548,640 by Medical Research Council describes CDRgrafting techniques and U.S. Pat. No. 5,859,205 by Celltech describesthe production of humanised antibodies. In vitro antibody libraries are,among others, disclosed in U.S. Pat. No. 6,300,064 by MorphoSys and U.S.Pat. No. 6,248,516 by MRC/Scripps/Stratagene. Phage Display techniquesare for example disclosed in U.S. Pat. No. 5,223,409 by Dyax. Transgenicmammal platforms are for example described in US200302048621 byTaconicArtemis.

The term “new antibody format” encompasses, for example bi- ortrispecific antibody constructs, Diabodies, Camelid Antibodies, DomainAntibodies, bivalent homodimers with two chains consisting of scFvs,IgAs (two IgG structures joined by a J chain and a secretory component),shark antibodies, antibodies consisting of new world primate frameworkplus non-new world primate CDR, dimerised constructs comprisingCH3+VL+VH, and antibody conjugates (e.g., antibody or fragments orderivatives linked to a toxin, a cytokine, a radioisotope or a label).This list is however not restrictive.

Further, the term also encompasses immunotoxins, i.e., heterodimericmolecules consisting of an antibody, or a fragment thereof, and acytotoxic, radioactive or apoptotic factor. Such type of format has forexample been developed by Philogen (e.g., anti-EDB human antibody L19,fused to human TNF), or Trastuzumab emtansine (T-DM1), which consists oftrastuzumab linked to the cytotoxoic Mertansine (DM1).

The term “fusion peptide” or “fusion protein” proteins relates, forexample, to proteins consisting of an immunoglobulin Fc portion plus atarget binding moiety (so-called -cept molecules).

In another preferred embodiment of the method according to theinvention, it is provided that the immunoligand has a reduced degree offucosylation compared to an immunoligand expressed in the absence of anelevated concentration of manganese or manganese ions.

Preferably, the degree of fucosylation is determined by methodsaccording to the art. Such methods comprise, among others, digestionwith Peptide-N-Glycosidase F (PNGase F), to deglycosylate the antibodies(see description at FIG. 1 for more details), and subsequent collectionof the isolated glycanes. The collected glycanes are labeled withanthranicilic acid and then analyzed by means of NP HPLC. Full detailsof the method are disclosed in Anumula (2012), content of which isincorporated herein by reference.

The term “absence of an elevated concentration of manganese or manganeseions.” means that during the process or in the preparation of theprocess, no manganese or manganese ions have willingly been introduced.This does not exclude that traces of manganese naturally occurring inmedia like water can still be present.

In a preferred embodiment of the method according to the invention, itis provided that the immunoligand demonstrates an increased ADCCactivity compared to an immunoligand (i) expressed in the absence of anelevated concentration of manganese or manganese ions or (ii) having ahigher degree of fucosylation.

The term “ADCC” relates to a mechanism of cell-mediated immune defensewhereby an effector cell of the immune system actively lyses a targetcell, whose membrane-surface antigens have been bound by specificantibodies. It is one of the mechanisms through which antibodies, aspart of the humoral immune response, can act to limit and containinfection. Classical ADCC is mediated by natural killer (NK) cells;macrophages, neutrophils and eosinophils can also mediate ADCC. ADCC ispart of the adaptive immune response due to its dependence on a priorantibody response.

Preferably, the ADCC activity is determined by methods according to theart. Such methods comprise, among others, the cytotoxicity assay asshown in FIG. 3.

Other suitable assays include chromium-51 [Cr51] release assay, europium[Eu] release assay, and sulfur-35 [S35] release assay. Usually, alabelled target cell line expressing a certain surface-exposed antigenis incubated with antibody specific for that antigen. After washing,effector cells expressing Fc receptor CD16 are co-incubated with theantibody-labelled target cells. Target cell lysis is subsequentlymeasured by release of intracellular label by a scintillation counter orspectrophotometry. The coupled bioluminescent method aCella TOX is nowin widespread use for ADCC and other cytotoxicity assessments. Sincethis technique measures the release of enzymes naturally present in thetarget cells, no labeling step is required and no radioactive agents areused.

Preferably, the immunoligand targets one or more cellular surfaceantigens involved in cell-mediated immune defense.

Preferably, said cellular surface antigens are selected from the groupconsisting of cyclophilin C, complement factor I, CD6, CD5, bovine WC-1and M130.

CD6 is an important cell surface protein predominantly expressed byhuman T cells and a subset of B cells, as well as by some B cell chroniclymphocytic leukemias and neurons (Aruffo et al. 1991, Kantoun et al.1981, Mayer et al. 1990). CD6 is a member of a large family of proteinscharacterized by having at least one domain homologous to the scavengerreceptor cysteine-rich domain (SRCR) of type I macrophages (Matsumoto etal. 1991 and Resnick et al. 1994). Other members of this family includeCD5 (Jones et al., 1986) cyclophilin C (Friedman et al. 1993),complement factor I, which binds activated complement proteins C3b andC4b (Goldberger, et al., J. Biol. Chem. 1987, 262:10065), bovine WC-1expressed by .tau./.delta. T cells (Wijingaard et al. 1992) and M130(Law et al. 1993), a macrophage activation marker.

Other preferred surface antigens encompass CD20, EGFR, HER2/neu, andmembrane-bound TNF.

In a preferred embodiment of the method according to the invention, itis provided that the immunoligand is Itolizumab.

Itolizumab (INN, trade name Alzumab®) is a ‘first in class’ humanizedIgG1 monoclonal antibody developed by Biocon. It selectively targetsCD6, a pan T cell marker involved in co-stimulation, adhesion andmaturation of T cells. Itolizumab, by binding to CD6, down regulates Tcell activation, causes reduction in synthesis of pro-inflammatorycytokines and possibly plays an important role by reducing T cellinfiltration at sites of inflammation. A double blind, placebocontrolled, phase III treat -Plaq study of itolizumab successfully metthe pre-specified primary end-point of significant improvement inPASI-75 (Psoriasis Area and Severity Index) score after 12 weeks oftreatment in patients with moderate to severe psoriasis compared toplacebo. Biocon received marketing authorization for the drug from theDrugs Controller General of India (DCGI) in January 2013 and marketingwithin India commenced in August 2013 (Jayaraman, 2013).

Itolizumab is produced from mouse derived NS0 cell line (called herein“T1h”) and also from Chinese Hamster Ovary (CHO) cell line (calledherein “Bmab-600”). The Fc portions of Bmab-600 and T1h bind to FcγRIIIawith different affinities as the post translational modifications,especially the afucosylation pattern varies with cell line and cultureconditions.

Itolizumab can for example be produced from mouse derived NS0 cell line(called herein “T1h”) and also from Chinese Hamster Ovary (CHO) cellline (called herein “Bmab-600”). The Fc portions of Bmab-600 and T1hbind to FcγRIIIa with different affinities as the post translationalmodifications, especially the afucosylation pattern varies with cellline and culture conditions. According to another aspect of theinvention, a glycoprotein is provided, which glycoprotein is producedwith a method or process according to any of the method of theinvention.

Preferably, said glycoprotein is a recombinant protein. More preferably,said glycoprotein is an immunoligand, preferably an antibody. It isparticularly preferred that said glycoprotein has a decreased fucosecontent in its glycosylation pattern.

Preferably, the glycoprotein, or a subdomain thereof, like an Fc region,has an afucosylation level of around 35%.

In a preferred embodiment, it is provided that the glycoprotein has anincreased ADCC. Preferably, said glycoprotein is Itolizumab.

In another preferred embodiment, it is provided that the glycoproteineffects in vitro- or in vivo reduction of cells being positive for CD25and CD4, in particular of CD4+ T cells.

The inventors have surprisingly shown that the use of anti-CD6 antibodyaccording to the invention leads to reduced proliferation of cells whichare positive for the surface antigens CD25 and CD4 (see FIG. 5B anddescription), in particular CD4+ T-Cells.

The term “reduction of cells”, as used herein, refers to (i) theinhibition of proliferation, (ii) the depletion, (iii) induction ofapoptosis or (iv) other mechanisms which lead to a reduction of suchcells.

According to another aspect of the invention, the use of a glycoproteinas set forth above for the manufacture of a medicament for the treatmentof a human or animal patient is provided. Likewise, the use of aglycoprotein as set forth above for the treatment of a human or animalpatient is provided.

In a preferred embodiment of such use, the human or animal patientsuffers from or has been diagnosed to be at risk to develop a diseaseselected from the group consisting of

-   Neoplastic diseases, including tumors, lymphomas and leukemias, in    particular B-cell chronic Lymphocytic leukemia (B-CLL), particularly    T-cell leukemias-   Autoimmune disease, including Rheumatoid arthritis, Psoriasis,    Crohn's disease, Lupus erythematosus, and/or Sjogren's disease-   Neurodegenerative diseases, including Multiple sclerosis, and/or    Parkinson's disease, Alzheimer's disease, Huntington's disease    and/or Amyotrophic lateral sclerosis, and/or-   Infectious diseases

Preferably, such use relates to the treatment or prevention of aversereactions like GVHD (Graft vs. Host disease) in a human or animal thathas been transplanted. Such transplantation includes organ transplantsas well as bone marrow transplants.

EXPERIMENTS AND FIGURES/EXAMPLES

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive; theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed invention, from a study ofthe drawings, the disclosure, and the appended claims. In the claims,the word “comprising” does not exclude other elements or steps, and theindefinite article “a” or “an” does not exclude a plurality. The merefact that certain measures are recited in mutually different dependentclaims does not indicate that a combination of these measures cannot beused to advantage. Any reference signs in the claims should not beconstrued as limiting the scope.

FIG. 1: Results of a deglycosylation experiment carried out with anantibody having an Fc region.

The anti CD antibody Itolizumab (also called T1h), has been incubatedwith a deglycosylation buffer (50 mM Tris, 1 mM CaCl2, pH=8.1) in a 1:1ratio to Itolizumab (5 mg/ml) followed by 24 hours incubation ofPeptide-N-Glycosidase F (PNGase) enzyme (10 U for 1 mg antibodies).

After incubating for 24 hrs at 37° C., an equal volume of T1h buffer(Histidine Trehalose buffer) in sample is added and centrifuged incentricon tubes (50 kD cut off filters) at 4° C., 4000 rpm for 15minutes. The residual volume is re-suspended again in equal volume withT1h buffer and centrifuged at 4° C., 4000 rpm for 15 minutes. Thede-glycosylated Ab is stored in final storage tube and concentrationestimated by Nano drop. The deglycoslation is confirmed by CE-SDS(Capillary Electrophoresis). FACS (Fluorescence activated cell sorter)analysis has then been carried out. Briefly, HUT78 cells (T cells line)are labelled with the anti CD6 antibody T1h, or the deglycosylated T1hantibody produced as described above.

Subsequently coming with a secondary anti Fc antibody, labelled withFITC, a signal is observed. FIG. 1 shows that deglycosylation of the Fcregion of Th1 does not compromise its ability to bind to CD6 expressingcell lines. These results have further been confirmed by Plasmonresonance experiments.

FIG. 2: Result of a deglycosylation experiment carried out with anantibody having an Fc region.

In particular, the anti CD antibody Itolizumab was deglycosylated asdiscussed supra. It's ability to inhibit of proliferation of activated Tcells was then compared with that of unmodified T1h in a suitableproliferation assay. Nimotuzumab, which is an antibody that has the sameIgG backbone as that of Itolizumab but binds to EGFR, was used asnegative control.

Briefly, the antibody was coated on sterile 96 well plates in aconcentration range 0-1 μg/ml overnight with bicarbonate buffer atpH9.5. After washes purified lymphocytes from normal healthy volunteerswere added to the plates. Itolizumab from 80-1 μg/ml was added and theculture was incubated for 4 days. Alamar blue was added to measureproliferation. Fold difference is calculated relative to unstimulatedcells control. Isotype Nimotuzumab antibody was used as control. Platebound anti CD3 (the anti CD3 used is OKT3 clone manufactured at centerfor molecular immunology, Cuba) stimulates the proliferation of naïve Tcells (Peripheral Blood Mononuclear Cells (PBMC) from a human donor,purified over a density gradient of Ficoll) from normal healthyvolunteers.

Nimotuzumab (80 μg/ml) does not show any inhibition of the T-Cellproliferation, resulting in about 2.75 fold increase in cells relativeto unstimulated cells, while native T1h shows inhibition of the T-Cellproliferation (35-20% inhibition in the 80 μg/ml-1.25 μg/ml range). Incontrast thereto, the impact of deglycosylated T1h is similar to that ofNimotuzumab. This means that upon deglycosylating, the antibody losesits ability to inhibit the proliferation of T cells.

FIG. 3: Results of a cytotoxicity assay comparing native antibody anddeglycosylated antibody.

Frozen PBMCs were thawed in RPMI 1640 Media with 10% FBS in presence ofIL-2 (Conc. 2.5 ng/mL) and incubated overnight in a 37° C., 5% CO₂incubator. On the next day cells were resuspended in media without IL-2and incubated for 4-5 hrs. In a 96 well plate 12,000 Hut-78 cells/50 μLwere added to each well. 50 μL 3× concentrated drug (either native T1h,deglycosylated T1h or anti CD3 at 10 microgram/ml) as per template wereadded and incubated for 2 hours at 37° C., 5% CO₂ incubator. PBMCs wereresuspended in assay media and 240,000 PBMCs/50 μL/well were added, toobtain a target to effector ratio of 1:20. The plates were incubated at37° C., 5% CO₂ incubator for 22 hours. 50 μL of Cyto Tox-Glo was addedto the plates and incubated for 30 minutes at room temperature. Theplates were read using Spectramax for luminescence to determine thecytotoxicity.

While native T1h shows mild but statistically consistent AntibodyDependent Cellular Cytotoxicity (ADCC) activity relative to anti CD3,which is a partially depleting antibody targeting T cells, this ADCCactivity is significantly reduced on deglycosylation of the molecule,indicating the effector function of T1h. Use of Fab2 fragment ofItolizumab can also reduce the ADCC activity comparable to thedeglycosylated molecule.

FIG. 4: Results of a Mixed Lymphocyte Reaction (MLR) experimentcomparing native antibody and deglycosylated antibody.

Preparation of PBMCs: 30 ml of blood was collected from a healthy donor.PBMCs were isolated by standard FICOLL density gradient centrifugationMonocyte Depletion & Setting up

Dendritic Cell (DC) Derivation Assay: These cells were incubated in aCO₂ incubator for two hours. Monocytes were allowed to adhere onto theplastic surface. The non-adhered cells (PBLs) were subsequently removedfrom the flasks. All the flasks were washed with 1×PBS once. 20 ml of DCmedia (made 50 ml stock, 10 μl of Granulocyte macrophagecolony-stimulating factor (GMCSF) and 5 μl of IL-4 in 50 ml of assaymedia) was added to each flask. The flasks were kept in CO₂ incubatorfor 6 days.

LPS Treatment to on-growing Dendritic Cells: At day 6, DC media with LPS(Lipopolysaccharides) was added to each flask (final concentration ofLPS in the flask is 4 ug/ml) and kept back in CO2 incubator for 40-48hrs.

Preparation of DCs: After LPS treatment the cell suspension (DC) wascollected from the two flasks. Each flask was washed with 1×PBS once.The cell suspension was spun down at 1500 rpm for 5 minutes andreconstituted in 3 ml media. LPS treated DCs were counted andreconstituted in media as per assay requirement.

Preparation of PBLs: Following the same protocol as mentioned before,Ficoll separation was performed after collecting blood from anotherhealthy individual. After monocyte depletion the non adhered Peripheralblood lymphocytes (PBLs) were collected and spun down at 1500 rpm for 5minutes and reconstituted in 5 ml media. PBLs were counted andreconstituted to 1.0×10⁶ cells/ml.

SEB treatment to Dendritic Cells (DC): Staphylococcal enterotoxin B(SEB) stock concentration is 1 mg/ml. From the stock 3 μl of SEB isdissolved in 3 ml of media to get 1 mg/ml working solution of SEB. Asper the standardized protocol 0.06×10⁶ DCs are treated with 0.6 ug ofSEB. A stock 0.1×106 cells/ml (LPS treated matured DCs) is made. Fromthis, 600 μl of cell suspension is dissolved in 2.4 ml of assay media(total volume of cell suspension is 3 ml that contains 0.02×10⁶cells/ml). This is spun down at 1500 rpm for 5 min and 600 ml of SEB (1ug/ml) is added to the pellet. This is incubated inside CO2 incubator at37° C. for 20 minutes. Excess media (2 ml) is added to the tube afterincubation and washed at 1500 rpm for 5 min. Supernatant is discardedand the cells are washed again with 3 ml of media. Finally the pellet isdissolved in 3 ml of assay media.

Mytomycin C treatment to PBLs: 25 μg/ml Mytomycin solution is made fromthe Mytomycin stock of 1 mg/ml. 0.5×10⁶ PBLs are treated with 500 μl of25 μg/ml Mytomycin for 30 min inside CO2 incubator at 37° C. Excessmedia (2 ml) is added to it after the incubation and the cells arewashed at 1500 rpm for 5 media. Supernatant is discarded and the cellsare washed again with 3 ml of media.

MLR Assay—Inhibition of Proliferation: MLR assay is performed atDC:PBL=1:50 ratio. Negative control used is Nimotuzumab. Native T1h wastested against a Fab2 version thereof which lacks the fully functionalFc region. After 6 days the plate is read with alamar blue using Bio-TekSynergy HT Gen5 plate reader.

While the intact antibody can inhibit the proliferation of T cellsinduced in this reaction, negative control Nimotuzumab with differentspecificity cannot. T1h without the Fc region cannot inhibit the T cellproliferation either, suggesting that the glycosylated Fc region alongwith Fab is critical for the inhibitory capacity of T1h in this assay. Asimilar effect has also been observed with the use of a deglycosylatedT1h thereby confirming the need of the glycosylation for the effectorfunction of T1h

FIG. 5a : Results of another Mixed Lymphocyte Reaction (MLR) experimentcomparing different immunomodulators.

The protocol is identical to FIG. 4. It is a mixed lymphocyte reaction.In addition to native T1h at four concentrations other immunosuppressantand immunomodulators were used, namely pimecrolimus (Pim), Abatacept(Aba) and Daclizumab (Dac) are included as positive controls for theassay. Nimotuzumab (hR3) is used as a negative control.

It turned out that T1h is able to reduce the proliferation of T cellsinduced in a mixed lymphocyte reaction as compared to an isotypeantibody, Nimotuzumab binding to Human EGFR. The fold reduction inducedby T1h is comparable to that induced by Abatacept (CTLA4-IgG1Fc),Daclizumab (Anti CD25) and Pimecrolimus (small molecule, IL2 inhibitor).

FIG. 5b : Analysis of the experiments shown in FIG. 5 a.

The analysis relates to cells from the culture after 144 hours (6 days)in the mixed lymphocyte reaction. B−−, B++, B+− and B−+ are thequadrants. Here the cells in culture in an MLR are evaluated after 6days. Although the inhibitory capacity of T1h compares well with otherantibodies, the path is different for T1h as here unlike with the othermolecules there is a significant decrease in CD4/CD25 activated T cellpopulation. T1h shows reduction in CD25+, CD4+ as well as CD4+ T cells.This indicates selective depletion of a subset of T cells. Hence,although as shown in FIG. 5A, the inhibition in the MLR by T1h iscomparable to that of Daclizumab, Abatacept and Pimecrolimus, only T1hshows a decrease in CD25+, CD4+ as well as CD4+ T cells.

FIG. 6: Results of a cytotoxicity assay comparing native antibody anddeglycosylated antibody.

The same assay in FIG. 3 was used to evaluate the antibodies withdifferent afucosylated content, compared to the positive control,anti-CD3. The data shown is a compilation from n=4 independentexperiments.

Afucosylation took place as described elsewhere herein (see, e.g.,description of FIG. 11). Increased afucosylation of the Fc region ofItolizumab shows a linear increase in the ADCC activity exhibited by theantibody with respect to the positive control antibody (anti-human CD3).This demonstrates the ability of Itolizumab to be more cytotoxic bymerely increasing the afucosylated Fc Glycan content. For example, toenhance the ADCC from 20% relative to that of anti CD3 to greater than40%, the afucosylated content in the antibody should be greater than10%.

Such increase may be caused by better binding to FcγRIII as shown in thebiacore data discussed infra (wherein Bmab 600 binds with betteraffinity as compared to T1h). Hence increasing the afucosylated speciesin the antibody can cause better binding to FcγRIII and this translatesinto a functional activity of better ADCC.

FIG. 7: Results of a CDC assay comparing T1h and Rituximab

The Human T cell lymphoma cell line Hut 78 (ATCC® TIB-161™), was used toassess the CDC activity of T1h. 1×10⁴ cells were incubated with therespective drug dilutions at 10 μg/mL, 1 μg/mL and 0.01 μg/mL for 20minutes in a 37° C., 5% CO2 incubator. Pooled normal human serum wasadded at a final concentration of 1:10 and cells were incubated for 2hours at 37° C. alamarBlue® (Invitrogen) was added and cells wereincubated for 20-22 hours at 37° C. The uptake of the dye by cells,followed by its reduction is read as fluorescence at 530/590 nm.Rituximab, an anti CD20 targeting CD20 receptors on a B cell line(Daudi) and causing complement-dependent cytotoxicity (CDC) was used a sa positive control in the assay to show that the serum componentsresulting in CDC was intact.

T1h does not exhibit CDC activity. Increase in the afucosylated speciesof Itolizumab does not increase the CDC activity of the molecule,concluding that only ADCC effector functions are enhanced with increasein afucosylation.

TABLE 1 Glycan profile of differentially afucosylated T1h samples usedin assays shown in FIGS. 6 and 7. Man6, Batch G0-GN G0f-GN G0 G0f Man5G1f-GN, G1 G1f (G1f-GN)S1 T1h Range 0.1-0.5 0.8-2.2 0.1-0.3 23.5-36.71.2-3.7 1.2-3.4 37.9-43.8 0.6-1.4 1185/12/03/25D 0.7 2.2 5.1 45.1 3.03.4 31.3 1.3 1185/12/03/27D 0.5 1.5 4.3 42.7 2.6 3.6 34.4 1.31185/12/03/34D 0.5 1.3 4 45.1 6.8 2.9 29.2 1.8 1185/12/03/31D 0.6 1.54.5 42.4 7.3 3.7 29.1 2.1 1185/11/01/35D 0.7 1.4 6.5 46.7 4.8 3.5 28.21.4 G1FS1, Tri G2fS1, Others Total non antennary complex with smallhybrid fucosylated Batch with 1G, hybridS1 G2f hybrid G2fS2 speciesspecies T1h Range 1.6-2.6 8.2-12.2 6.5-13.6 1.5-3.8 0.5-1.3 3.951185/12/03/25D 1.0 4.2 1.9 0.2 0.7 10.1 1185/12/03/27D 1.0 5.0 2.1 0.10.8 8.7 1185/12/03/34D 0.9 4.2 1.9 0.7 0.9 13.1 1185/12/03/31D 1.0 4.02.3 0.3 1.1 14.5 1185/11/01/35D 0.8 3.4 1.3 0.6 0.7 13.4

The analysis of Glycosylation patterns took place with standard methods.In brief, the antibodies were digested with Peptide-N-Glycosidase F(PNGase F), to deglycosylate the antibodies (see description at FIG. 1for more details), and the isolated glycanes were collected. Thecollected glycanes were labeled with anthranicilic acid and thenanalyzed by means of NP HPLC. Full details of the method are disclosedin Anumula (2012), content of which is incorporated herein by reference.

In this table, the following abbreviations are used: G0=no Galactose,G1=1 terminal Galactose residue, G2=2 terminal Galactose residues,GN=N-Acetyl Glucosamine or GlcNac, F=Fucose, Man5=5 mannose residues,Man6=6 mannose residues and S=Sialic acid.

An explanation of the Glycosylation patterns determined in the course ofthe experiments shown herein, and the nomenclature used, is provided inFIG. 15.

FIGS. 8-10: Binding curves of T1h to FcγRIIIa detect with Plasmonresonance

BIAcore is an analytical device which detects differences in surfaceplasmon resonance-based changes in the refractive index near a sensorsurface. This method of determining affinity constants of an antibodyfor Fc receptors ligands has been used widely. In order to detect aninteraction one molecule (the ligand) is immobilized onto the sensorsurface. Its binding partner (the analyte) is injected in aqueoussolution (sample buffer) through the flow cell, also under continuousflow. As the analyte binds to the ligand, the accumulation of protein onthe surface results in an increase in the refractive index, which isplotted against time to yield a sensorgram. Association (K_(a)),dissociation-rate constants (K_(d)) and equilibrium dissociationconstants (K_(D)) are determined from the analysis of sensorgrams.

FcγRIIIa is considered as an intermediate affinity receptor. It canvariably bind monomeric IgG and appears to have a high affinity for IgGthan the lower affinity Fc gamma receptors. They are expressed on the NKcells and monocytes of the blood cells.

The Fc portions of Bmab-600 and T1h bind to FcγRIIIa with differentaffinities as the post translational modifications, especially theafucosylation pattern varies with cell line and culture conditions. Weevaluated these two products binding affinities towards FcγRIIIa inBiacore instrument. The binding affinity results of Bmab-600 show higheraffinity in binding to FcγRIIIa receptors in comparison to T1h. Thefollowing samples were analyzed on the surface immobilized with FcγRIIIareceptor:

1. T1h antibody2. Bmab-600 antibody3. Deglycosylated T1h antibody

Each sample was analyzed two times and the average K_(D) (μM) values arereported and compared against each other. FIG. 8 shows the bindingcurves of T1h antibody to FcγRIIIa, FIG. 9 shows the binding curves ofBmab-600 antibody to FcγRIIIa, and FIG. 10 shows the binding curves ofdeglycosylated T1h antibody to FcγRIIIa.

The method was sensitive and was able to pick-up the differences betweenafucosylation differences that were existing inherently in thedifferentially afucosylated samples of Bmab-600 and T1h. The data alsoshows that as the afucosylation levels increases the FcγRIIIa bindingaffinity values decreases (meaning higher affinity) proportionally. Themethod specificity was also demonstrated by analyzing the deglycosylatedsample of T1h where no binding interactions was observed.

TABLE 2 Kinetic values of T1h antibody to FcγRIIIa (see FIG. 8) ka kdK_(D) K_(D) Average K_(D) Samples (1/Ms) (1/s) (M) (μM) (μM) T1h1.52E+04 5.65E−03 3.72E−07 0.372 0.440 (NS0) 1.16E+04 5.88E−03 5.08E−070.508

TABLE 3 Rate constant values of Bmab600 antibody to FcγRIIIa (see FIG.9) ka kd K_(D) K_(D) Average K_(D) Samples (1/Ms) (1/s) (M) (μM) (μM)Bmab- 2.46E+04 4.76E−03 1.93E−07 0.193 600 2.78E+04 5.73E−03 2.07E−070.207 0.200 (CHO)

TABLE 4 Rate constant values of deglycosylated T1h antibody to FcγRIIIa(see FIG. 10) ka kd K_(D) K_(D) Average K_(D) Samples (1/Ms) (1/s) (M)(μM) (μM) T1h (NS0) Negative interaction

TABLE 5 Rate constant values of differentially afucosylated samples ofBmab-600 and T1h antibody to FcγRIIIa (see FIG. 10) % afu- ka kd K_(D)K_(D) Samples cosylation (1/Ms) (1/s) (M) (μM) T1h 2.5 1.18E+04 5.54E−034.686E−07 0.468 Bmab-600 5.1 1.28E+04 4.20E−03 3.28E−07 0.328 Bmab-6009.6 1.96E+04 6.00E−03 3.06E−07 0.306 Bmab-600 35.6 2.73E+04 5.34E−031.95E−07 0.195

FIG. 11: Afucosylation caused by addition of manganese (Mn)

Addition of Mn at concentrations higher than the media concentration(0.005 μM) was tested for a CHO-S cell line producing T1h monoclonalantibody. The trials were started with initial cell count of 0.8-0.9million cells/ml. Regular feeding of glucose and amino acids was carriedout during the process to meet the nutritional requirement of cells.Periodic samples were taken to check the cell growth, viability and IgGtitre profiles. The broths were harvested at the end of the culture andanalyzed for glycosylation profiles as described elsewhere herein.

The trials were done in 2 sets. The first set was carried out in shakeflasks and the second set was performed in 50 L bioreactors. Manganesewas added in culture medium and through feed at specified intervalsduring the run.

FIG. 11 shows an increase in afucosylation level with addition ofManganese in the culture medium and through feed. The afucosylationprofiles correspond to day 10 sample in case of 0.1 mM, 0.2 mM and 0.25mM; and day 11 in case of 0.075 mM and 0.23 mM. The results aresummarized in the following tables:

TABLE 6 Glycan profile of a 10-day shake flask trial with 0.1 mM, 0.2 mMand 0.25 mM Manganese (Mn) concentrations Mn concentration tested G0-GNG0f-GN G0 G0f Man5 G1f-GN, G1 G1f

0.1 mM 0.5 1.1 4.9 56.7 3.1 1.9 25.2 1.1 0.2 mM 0.6 0.9 6.3 55.1 4.2 2.324.1 1.2 0.25 mM  1.2 1 10.3 51.2 6.8 3.1 20.3 1.1 G1FS1, Tri antennarycomplex G2fS1, Other with 1 G,

with small hybrid Mn concentration tested

G2f hybrid G2fS2 species Afucosylation 0.1 mM 0.8 2.5 1.2 0.5 0.5 9.60.2 mM 0.8 2.4 1.1 0.5 0.7 12.3 0.25 mM  0.7 1.9 1 0.4 1 19.4

indicates data missing or illegible when filed

TABLE 7 Glycan profile of 50 L batches run for 11 days with 0.075 mM and0.23 mM Mn concentrations Man6, Mn concentration tested G0f-GN G0 G0fMan5 G1f-GN, G1 G1f (G1f-GN)S1 0.075 mM

1 2 40.6 1.9 2.3 38.9 1.8  0.23 mM

1.5 4.5 42.4 7.3 3.7 29.1 2.1 G1FS1, Tri G2fS1, Other antennary complexwith small hybrid Mn concentration tested with 1 G,

G2f hybrid G2fS2 species Afucosylation 0.075 mM 1.1 6.2 2.3 0.9 0.8 5.9 0.23 mM 1 4 2.3 0.3 1.1 14.5

indicates data missing or illegible when filed

Based on the above experiments, an increase in % afucosylation wasobserved with increase in total manganese concentration. The cellgrowth, viability and IgG titre profiles were not affected by Mnaddition.

FIG. 12: Increase in G0, Man5 and afucosylation levels by addition ofmanganese (Mn)

The Effect of manganese in the range of 0.0025 μM to 0.5 mM was testedby varying the concentration in culture medium. No manganese additionwas done through feeds. The trial was carried out in shake flasks andsamples were analyzed for glycosylation profiles on day 8. A gradualincrease in G0, Man5 and afucosylation levels with an increase inmanganese concentration could be observed.

FIG. 13: Copper concentration does not have an effect on fucosylation.

To evaluate the effect of other divalent cations, Cu was selected forthe study since Cu was also a co-factor in the glycosylation pathway(for enzyme Sialyltransferase). Different concentrations of copper inculture medium in the range of 0.01 μM to 200 μM were tested in shakeflasks. No increase/effect in any of the values (G0, Man5 andafucosylation) was observed, as shown in FIG. 3. This establishes thatcopper ions does not affect afucosylation levels in proteins.

FIG. 14: Schematic representation of an immunoglobulin G

FIG. 14 shows a schematic representation of an immunoglobulin G (IgG).An IgG is composed of two identical light chains (each composed of twodomains, V_(L) and V_(H)) and two identical heavy chains (each composedof four domains, V_(H), C_(H)1, C_(H)2 and C_(H)3). Antigen bindingsurface is formed by the variable domains of heavy and light chains andthe effector function, such as complement activation and binding ofcytotoxic cells is mediated by the Vc region of the antibody.

FIG. 15: Nomenclature of N-glycan structures

FIG. 15 shows an overview of different N-glycans. Generally, the term“N-glycosylation” refers to glycosylation of the amino acid residueasparagine (N). Here, an oligosaccharide chain is attached byoligosaccharyltransferase to those asparagine residues which occur inthe tripeptide sequences Asn-X-Ser or Asn-X-Thr, where X can be anyamino acid except Pro.

The experiments shown herein clearly demonstrate that

-   a) the fucose content of glycoproteins can be manipulated by varying    the total concentration of manganese or manganese ions in media and    feeds in the protein expression process-   b) increasing total concentration of manganese or manganese ions    leads to an increased afucosylation, or to a decreased fucose    content in the glycosylation pattern of glycoproteins.-   c) in immunoligands like antibodies having an Fc region, protein    expression in the presence of an elevated concentration of manganese    or manganese ions leads to (i) a higher degree of afucosylation    and (ii) an increased ADCC-   d) in these immunoligands, increasing the degree of afucosylation    does not lead to an increased CDC-   e) deglycosylation of immunoligands like antibodies having an Fc    region, by contrast, does not lead to an increased ADCC-   f) other than afucosylation, deglycosylation of immunoligands like    antibodies having an Fc region can lead to loss of functional    activity of such immunoligands, in particular if such functional    activity is related with activity like effector function and/or    ADCC.

REFERENCES

-   Jayaraman K, Nature Biotechnology 31, 1062-1063 (2013)-   Anumula K R, Glycobiology (2012) 22 (7): 912-917.-   Shields et al, J Biol Chem 277:26733-26740.-   Konno et al, Cytotechnology. 2012 May; 64(3):249-65-   Aruffo et al., J. Exp. Med. 1991, 174:949-   Kantoun et al., J. Immunol. 1981, 127:987-   Mayer et al., J. Neuroimmunol. 1990. 29:193-   Matsumoto, et al., J. Exp. Med. 1991, 173:55-   Resnick et al., Trends Biochem. Sci. 1994, 19:5-   Jones et al., Nature. 1986, 323:346-   Friedman et al. 1993, PNAS 90:6815-   Goldberger, et al., J. Biol. Chem. 1987, 262:10065-   Wijingaard et al., J. Immunol. 1992, 149:3273-   Law et al., Eur J. Immunol. 1993, 23:2320

1. A method for modifying fucosylation in a eukaryote and/or aeukaryotic protein expression system, the method comprising controllingthe total concentration of manganese or manganese ions in the medium. 2.The method according to claim 1, wherein the method decreasesfucosylation when expression of a protein and/or post-translational iscarried out modification in a eukaryotic protein expression system andin the presence of an elevated total concentration of manganese ormanganese ions.
 3. The method according to claim 2, wherein the elevatedconcentration of manganese or manganese ions is in the range of ≧0.05mM-≦10 mM.
 4. The method according to claim 2, wherein the proteinexpression and/or post-translational modification is carried out in aprotein expression system selected from the group consisting of Insectcells Fungal cells Yeast cells Protozoan cells, and/or Mammalian cells5. The method according to claim 2, wherein the protein is aglycoprotein.
 6. The method according to claim 2, wherein the protein isa recombinant protein.
 7. The method according to claim 5, wherein theglycoprotein is an immunoligand.
 8. The method according to claim 7,wherein the immunologand is at least one selected from the groupconsisting of a monoclonal antibody and a fusion peptide consisting ofan immunoglobulin Fc region fused to a target binding moiety.
 9. Themethod according to claim 7, wherein the immunoligand has a reduceddegree of fucosylation compared to an immunoligand expressed in theabsence of an elevated concentration of manganese or manganese ions. 10.The method according to claim 7, wherein the immunoligand demonstratesan increased ADCC activity compared to an immunoligand (i) expressed inthe absence of an elevated concentration of manganese or manganese ionsor (ii) having a higher degree of fucosylation.
 11. The method accordingto claim 7, wherein the immunoligand targets one or more cellularsurface antigens involved in cell-mediated immune defense.
 12. Themethod according to claim 7, wherein the immunoligand is Itolizumab. 13.A glycoprotein or subdomain thereof, produced with the method of claim5.
 14. The glycoprotein according to claim 13, which glycoprotein is arecombinant protein.
 15. The glycoprotein according to claim 13, whichglycoprotein is an antibody.
 16. The glycoprotein according to claim 13,having a decreased fucose content in its glycosylation pattern.
 17. Theglycoprotein according to claim 16, having a fucosylation level ofaround 35%.
 18. The glycoprotein according to claim 16, having anincreased ADCC.
 19. The glycoprotein according to claim 16, wherein theglycoprotein is Itolizumab.
 20. The glycoprotein according to claim 19,having an effect of an in vitro- or in vivo reduction of cells beingpositive for CD25 and CD4. 21.-24. (canceled)